Fosfomycin is a safe, broad-spectrum antibiotic that is effective against both Gram-negative and Gram-positive bacteria. It inhibits cell wall biogenesis by covalently inactivating MurA, the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis. Unfortunately, shortly after the introduction of fosfomycin, bacterial strains resistant to the antibiotic began to emerge. Three types of closely related fosfomycin resistance enzymes are known: FosA, a fosfomycin-specific glutathione transferase;FosB, a fosfomycin-specific cysteine transferase;and FosX, a fosfomycin hydrolase. Recently, sequence analysis and biochemical studies revealed a FosX enzyme in Mesorhizobium loti (FosXMl) that demonstrates both FosA and FosX activities, but neither of these activities was substantial enough to confer fosfomycin resistance in vivo. It is hypothesized that this enzyme is involved in phosphonate metabolism and represents an evolutionary precursor to true fosfomycin resistance enzymes. This enzyme, therefore, presents a remarkable opportunity to explore how functional fosfomycin resistance enzymes might arise from a progenitor enzyme involved in metabolism. The overall goal of the proposed research is to identify which structural and functional elements are necessary for the evolution of an efficient fosfomycin resistance enzyme. In vitro DNA recombination and in vivo selection methods will be used together to produce efficient fosfomycin resistance enzymes from the FosXMl progenitor. Sequence analysis of recombinant enzymes will reveal what genetic material is transferred during recombination, and steady-state kinetic experiments and x-ray crystallographic analysis will be utilized to characterize the recombinant enzymes. The proposed research will give tremendous insight into how bacterial resistance to fosfomycin evolved from an enzyme involved in phosphonate metabolism. The conclusions from this study may provide direction for the discovery and design of inhibitors of fosfomycin resistance enzymes. PUBLIC HEALTH RELEVANCE: Microbial resistance to antibiotic compounds is a growing problem that represents a serious hazard to human health. The proposed research seeks to understand how enzymes that confer resistance to the antibiotic fosfomycin have evolved. An understanding of the key changes that occur during the evolution of these enzymes will help explain how resistance to antibiotics arises and may provide direction for the development of new therapeutic compounds to combat fosfomycin resistance.